This project involves the molecular genetic analysis of a set of interacting regulatory mechanisms in bacteriophage P22. Most of the genes of phage P22 are subject to negative control by the P22 c2 repressor (product of gene c2). The P22 ant gene codes for an antirepressor protein which interacts with and inhibits the c2 repressor protein. Synthesis of antirepressor is in turn under negative control by two repressors, the products of P22 genes arc and mnt. We have identified the arc gene product by SDS-polacrylamide gel electrophoresis of extracts of cells infected with P22 arc plus and arc-amber phage. Since we have been unable to detect the mnt gene product in this way, we will clone the mnt gene under control of the E. coli lac promoter in order to achieve high-level synthesis of Mnt. If we can locate the Mnt protein on gels, we can carry out experiments to determine how the mnt gene is expressed and regulated. We are also investigating how the sieA gene, which lies to the left of mnt, is expressed; we have evidence that mnt and sieA are not transcribed from the same promoter. The Arc and Mnt proteins probably work by preventing transcription from P ant. We have identified the P ant transcript produced in vitro using P22 DNA as template and determined the position of the ends of this RNA with respect to the restricton map of P22 DNA. We plan to isolate the P ant transcript produced in vivo and compare its rate of synthesis in the presence and absence of Mnt and Arc. We also plan to sequence the ends of both the in vivo and in vitro RNA's.